Eukaryotes maintain an array of subcellular organelles to spatially organize and sequester DNA, RNA, and proteins. Despite their relative lack of compartmentalization, bacteria have also developed mechanisms for protein localization. Proper localization is essential for a number of processes including chemotaxis, cell division, and adhesion. Our lab has recently published the protein localisome of C. crescentus which identified 300 proteins with distinct localizations. Three proteins were found to localize in the stalk of Caulobacter. This small subset of proteins is an excellent starting point for investigating the mechanisms of protein localization in bacteria. The aims of this proposal are to identify A) the trans-acting and B) cis-acting protein domains required for stalk localization. Initial experiments will determine whether stalk proteins are delivered directly to the stalk-pole or arrive by a diffusion and capture mechanism. Additional experiments will examine whether peptidoglycan or phospholipid binding is required for localization. Bacterial cell shape regulates protein localization, therefore a stalk anchor protein likely contains 2 domains: a) either a peptidoglycan or a phospholipid binding domain to allow binding to the stalk pole and b) a protein-protein interaction domain to bind the stalk proteins. The 3 stalk proteins have an SH3-ligand PxxP motif which, in eukaryotes, has been extensively studied as a protein-protein interaction domain. Correspondingly, there are 2 putative SH3- domain proteins in C. crescentus which may regulate localization via protein-protein interactions. Analysis of the C. crescentus genome has identified 9 potential peptidoglycan binding proteins which may play a role in stalk localization. These candidate proteins and motifs will be evaluated for their ability to regulate localization. In the event that these candidates do not regulate localization, I have proposed a set of assays that take advantage of our lab's expertise in high-throughput cloning and microscopy to perform large scale protein-interaction and genetic screens for localization regulatory proteins. PUBLIC HEALTH RELEVANCE: The experimental paradigm developed for this proposal has broad long-term applications. The wealth of data in our localisome will allow us to expand this project to identify localization mechanism for other subcellular regions such as the pole and the midcell. It is likely that these mechanisms will be broadly applicable in various bacterial species due to the conservation of basic proliferation mechanisms. Finally, mechanistic insight into protein localization will be of great clinical value. Since proper protein localization is required for bacterial proliferation, drugs that target localization may be a new source of antimicrobials to fight the growing problem of drug-resistance.